This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of a gene, pbp-nv2, encoding a novel high molecular weight penicillin binding protein (PBP), PBP-Nv2, from Streptococcus pneumoniae and the use of the gene and its encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.
The emergence of antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. The emergence and rapid spread of beta-lactam resistance in Streptococcus pneumoniae has been particularly problematic. This organism is responsible for many respiratory tract infections, and resistance to beta-lactam drugs has been attributed to a modification of one or more of the penicillin-binding proteins (PBPs). Furthermore, penicillin-resistant Streptococcus pneumoniae are frequently resistant to other commonly used antibiotics, such as erythromycin. These multi-drug resistant (MDR) organisms are a real threat to humans, particularly children and the elderly. Increasingly, the only drug that can be used to treat infections with MDR organisms is vancomycin, and there is considerable concern that the bacteria could also develop resistance to vancomycin.
The PBPs are involved in bacterial cell wall synthesis. The cell wall comprises a peptidoglycan layer which provides mechanical rigidity for the bacterium. The peptidoglycan layer is composed of a sugar backbone (alternating residues of N-acetylglucosamine and N-acetylmuramic acid) attached to a pentapeptide (also referred to as xe2x80x9cstem peptidexe2x80x9d) containing D and L amino acid residues. In the formation of the mature peptidoglycan, a lipid-linked disaccharide-pentapeptide is translocated across the cytoplasmic membrane, exposing the pentapeptide sidechains to the cell surface. Transglycosylation of the sugar residues then leads to polymerization of the backbone sugar residues. Further stabilization of the nascent peptidoglycan occurs by a transpeptidation enzymatic reaction that crosslinks adjacent pentapeptide moieties. The high molecular weight PBPs catalyze these final steps in peptidoglycan synthesis. Without the crosslinking step the peptidoglycan structure is severely weakened and susceptible to degradation. Indeed, the crosslinking step constitutes the target of action for antibiotic compounds such as penicillin and other beta-lactam drugs.
When effective as antibiotic agents, beta-lactam drugs interact with PBPs to form an acyl-enzyme intermediate. This intermediate is resistant to hydrolysis. Mechanistically, beta-lactam drugs act as irreversible inhibitors. Resistance to beta-lactam drugs in Streptococcus pneumoniae arises through mutation events such that one or more low-affinity xe2x80x9cmosaicxe2x80x9d PBPs replace a wild-type PBP. The molecular basis of resistance has in a few cases been correlated with specific mutations within a PBP gene. The discovery of new antibacterial compounds against the transpeptidase domain of PBP-Nv2 or to an unexploited target (e.g. the transglycosylase domain) would be particularly useful against Streptococcus pneumoniae infections.
The present invention is designed to meet the aforementioned need and provides, inter alia, isolated nucleic acid molecules that encode a novel PBP from Streptococcus pneumoniae. The invention also provides protein products encoded by the gene, in substantially purified form.
Having the cloned pbp-nv2 gene of Streptococcus pneumoniae enables the production of recombinant PBP-Nv2 protein and derivatives thereof for the implementation of assays and screens to identify new inhibitory compounds targeted at the peptidoglycan biosynthetic pathway.
In one embodiment the present invention relates to the isolated gene pbp-nv2 that encodes a novel Streptococcus pneumoniae PBP, PBP-Nv2,. The gene comprising the nucleotide sequence is identified as SEQ ID NO. 1.
In another embodiment the present invention relates to a novel protein molecule, PBP-Nv2, wherein said protein molecule comprises the sequence identified as SEQ ID NO. 2.
In another embodiment, the present invention relates to a soluble form of PBP-Nv2 (designated PBP-Nv2S) wherein PBP-Nv2S comprises amino acid residues 85 through 821, inclusive, of SEQ ID NO.2.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding PBP-Nv2 protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO. 3:
In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the Streptococcus pneumoniae pbp-nv2 gene in operable linkage to gene expression sequences enabling said PBP gene to be transcribed and translated in a host cell.
In still another embodiment the present invention relates to homologous or heterologous host cells that have been transformed or transfected with a vector carrying the cloned pbp-nv2 gene from Streptococcus pneumoniae such that said gene is expressed in the host cell.
In a still further embodiment, the present invention relates to a method for identifying compounds that bind the Streptococcus pneumoniae PBP-Nv2 protein or fragment thereof.